Search

Background: Phylogenetic mapping of HIV-1 lineages circulating across defined geographical locations is promising for better understanding HIV transmission networks to design optimal prevention interventions., Methods: We obtained near full-length HIV-1 genome sequences from people living with HIV (PLWH), including participants on antiretroviral treatment in the Botswana Combination Prevention Project, conducted in 30 Botswana communities in 2013-2018. Phylogenetic relationships among viral sequences were estimated by maximum likelihood., Results: We obtained 6078 near full-length HIV-1C genome sequences from 6075 PLWH. We identified 984 phylogenetically distinct HIV-1 lineages (molecular HIV clusters) circulating in Botswana by mid-2018, with 2-27 members per cluster. Of these, dyads accounted for 62%, approximately 32% (n = 316) were found in single communities, and 68% (n = 668) were spread across multiple communities. Men in clusters were approximately 3 years older than women (median age 42 years, vs 39 years; P < .0001). In 65% of clusters, men were older than women, while in 35% of clusters women were older than men. The majority of identified viral lineages were spread across multiple communities., Conclusions: A large number of circulating phylogenetically distinct HIV-1C lineages (molecular HIV clusters) suggests highly diversified HIV transmission networks across Botswana communities by 2018., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A national ART program was launched in Tanzania in October 2004. Due to the existence of multiple HIV-1 subtypes and recombinant viruses co-circulating in Tanzania, it is important to monitor rates of drug resistance. The present study determined the prevalence of HIV-1 drug resistance mutations among ART-naive female bar and hotel workers, a high-risk population for HIV-1 infection in Moshi, Tanzania. A partial HIV-1 pol gene was analyzed by single-genome amplification and sequencing in 45 subjects (622 pol sequences total; median number of sequences per subject, 13; IQR 5-20) in samples collected in 2005. The prevalence of HIV-1 subtypes A1, C, and D, and inter-subtype recombinant viruses, was 36%, 29%, 9% and 27%, respectively. Thirteen different recombination patterns included D/A1/D, C/A1, A1/C/A1, A1/U/A1, C/U/A1, C/A1, U/D/U, D/A1/D, A1/C, A1/C, A2/C/A2, CRF10&#95;CD/C/CRF10&#95;CD and CRF35&#95;AD/A1/CRF35&#95;AD. CRF35&#95;AD was identified in Tanzania for the first time. All recombinant viruses in this study were unique, suggesting ongoing recombination processes among circulating HIV-1 variants. The prevalence of multiple infections in this population was 16% (n = 7). Primary HIV-1 drug resistance mutations to RT inhibitors were identified in three (7%) subjects (K65R plus Y181C; N60D; and V106M). In some subjects, polymorphisms were observed at the RT positions 41, 69, 75, 98, 101, 179, 190, and 215. Secondary mutations associated with NNRTIs were observed at the RT positions 90 (7%) and 138 (6%). In the protease gene, three subjects (7%) had M46I/L mutations. All subjects in this study had HIV-1 subtype-specific natural polymorphisms at positions 36, 69, 89 and 93 that are associated with drug resistance in HIV-1 subtype B. These results suggested that HIV-1 drug resistance mutations and natural polymorphisms existed in this population before the initiation of the national ART program. With increasing use of ARV, these results highlight the importance of drug resistance monitoring in Tanzania.

Background: Determining patterns of HIV transmission is increasingly important for the most efficient use of modern prevention interventions. HIV phylogeny can provide a better understanding of the mechanisms underlying HIV transmission networks in communities., Methods: To reconstruct the structure and dynamics of a local HIV/AIDS epidemic, the phylogenetic relatedness of HIV-1 subtype C env sequences obtained from 785 HIV-infected community residents in the northeastern sector of Mochudi, Botswana, during 2010-2013 was estimated. The genotyping coverage was estimated at 44%. Clusters were defined based on relatedness of HIV-1C env sequences and bootstrap support of splits., Results: The overall proportion of clustered HIV-1C env sequences was 19.1% (95% CI 17.5% to 20.8%). The proportion of clustered sequences from Mochudi was significantly higher than the proportion of non-Mochudi sequences that clustered, 27.0% vs. 14.7% (p = 5.8E-12; Fisher exact test). The majority of clustered Mochudi sequences (90.1%; 95% CI 85.1% to 93.6%) were found in the Mochudi-unique clusters. None of the sequences from Mochudi clustered with any of the 1,244 non-Botswana HIV-1C sequences. At least 83 distinct HIV-1C variants, or chains of HIV transmission, in Mochudi were enumerated, and their sequence signatures were reconstructed. Seven of 20 genotyped seroconverters were found in 7 distinct clusters., Conclusions: The study provides essential characteristics of the HIV transmission network in a community in Botswana, suggests the importance of high sampling coverage, and highlights the need for broad HIV genotyping to determine the spread of community-unique and community-mixed viral variants circulating in local epidemics. The proposed methodology of cluster analysis enumerates circulating HIV variants and can work well for surveillance of HIV transmission networks. HIV genotyping at the community level can help to optimize and balance HIV prevention strategies in trials and combined intervention packages.

The study estimated the prevalence of HIV-1 intra-subtype recombinant variants among female bar and hotel workers in Tanzania. While intra-subtype recombination occurs in HIV-1, it is generally underestimated. HIV-1 env gp120 V1-C5 quasispecies from 45 subjects were generated by single-genome amplification and sequencing (median (IQR) of 38 (28-50) sequences per subject). Recombination analysis was performed using seven methods implemented within the recombination detection program version 3, RDP3. HIV-1 sequences were considered recombinant if recombination signals were detected by at least three methods with p-values of ≤0.05 after Bonferroni correction for multiple comparisons. HIV-1 in 38 (84%) subjects showed evidence for intra-subtype recombination including 22 with HIV-1 subtype A1, 13 with HIV-1 subtype C, and 3 with HIV-1 subtype D. The distribution of intra-patient recombination breakpoints suggested ongoing recombination and showed selective enrichment of recombinant variants in 23 (60%) subjects. The number of subjects with evidence of intra-subtype recombination increased from 29 (69%) to 36 (82%) over one year of follow-up, although the increase did not reach statistical significance. Adjustment for intra-subtype recombination is important for the analysis of multiplicity of HIV infection. This is the first report of high prevalence of intra-subtype recombination in the HIV/AIDS epidemic in Tanzania, a region where multiple HIV-1 subtypes co-circulate. HIV-1 intra-subtype recombination increases viral diversity and presents additional challenges for HIV-1 vaccine design.

Human immunodeficiency virus type 1 (HIV-1) Tat is a mediator of viral transcription and is involved in the control of virus replication. However, associations between HIV-1 Tat diversity and functional effects during primary HIV-1 infection are still unclear. We estimated selection pressures in tat exon 1 using the mixed-effects model of evolution with 672 viral sequences generated from 20 patients infected with HIV-1 subtype C (HIV-1C) over 500 days postseroconversion. tat exon 1 residues 3, 4, 21, 24, 29, 39, and 68 were under positive selection, and we established that specific amino acid signature patterns were apparent in primary HIV-1C infection compared with chronic infection. We assessed the impact of these mutations on long terminal repeat (LTR) activity and found that Tat activity was negatively affected by the Ala(21) substitution identified in 13/20 (65%) of patients, which reduced LTR activity by 88% (± 1%) (P < 0.001). The greatest increase in Tat activity was seen with the Gln(35)/Lys(39) double mutant that resulted in an additional 49% (± 14%) production of LTR-driven luciferase (P = 0.012). There was a moderate positive correlation between Tat-mediated LTR activity and HIV-1 RNA in plasma (P = 0.026; r = 0.400) after 180 days postseroconversion that was reduced by 500 days postseroconversion (P = 0.043; r = 0.266). Although Tat activation of the LTR is not a strong predictor of these clinical variables, there are significant linear relationships between Tat transactivation and patients' plasma viral loads and CD4 counts, highlighting the complex interplay between Tat mutations in early HIV-1C infection.

This study analyzed the distribution and prevalence of HIV-1 subtypes, multiplicity of HIV-1 infection, and frequency of inter-subtype recombination among HIV-1-infected female bar and hotel workers in Moshi, Kilimanjaro Region, Tanzania, from 2004 to 2007. The HIV-1 viral sequences spanning the V1-C5 region of HIV-1 env gp120 were analyzed from 50 subjects by single genome amplification and sequencing (SGA/S) technique. A total of 1740 sequences were amplified and sequenced from the HIV-1 proviral DNA template. The median env sequences analyzed per subject per two time points was 38 (IQR 28-50) over one year of HIV infection. In a subset of 14 subjects, a total of 239 sequences were obtained from HIV-1 RNA template at the baseline visit. The most prevalent HIV-1 subtypes were A1 (56%) and C (30%), while HIV-1 subtype D and inter-subtype recombinant viruses were found in 6% and 8% of subjects respectively. Transmission of multiple HIV-1 variants was evident in 27% of the subjects infected with pure HIV-1 subtypes A1, C, or D. The HIV-1 inter-subtype recombinants were found in 8% including HIV-1 C/A, D/A, and complex mosaic recombinants. Multiple viral variants were found in two subjects infected with inter-subtype recombinants. One subject harbored quasispecies of both pure HIV-1 A1 and C/A recombinant. The other subject was infected with two complex mosaic inter-subtype recombinant variants belonging to subtype D. HIV-1 multiple infections and ongoing recombination contribute significantly to the genetic diversity of circulating HIV-1 in Tanzania and have important implications for vaccine design and the development of therapeutic strategies.

Two analyses of HIV-1 subtype C Gag quasispecies were performed in a prospective cohort of 42 acutely and recently infected individuals by SGA on viral RNA/proviral DNA templates. First, in vivo Gag substitutions were assessed in relation to the HIV-1C consensus sequence, which revealed that 29.3% of detected amino acid substitutions can be classified as reversions to subtype consensus, 61.3% as forward substitutions from subtype consensus, and 9.3% as polymorphisms not associated with the subtype consensus sequence. Second, the proportion, dynamics, and relationships within individual pools of viral quasispecies were analyzed. Among reverse substitutions, 16.1% were minor, 11.0% transient, 13.6% dominant, and 59.2% fixed. In contrast, 31.6% of forward substitutions were minor, 59.3% transient, 3.8% dominant, and 5.3% fixed. The distinct patterns in the spectrum and dynamics of reverse and forward Gag substitutions suggest that these differences should be considered in HIV-1 evolutionary studies and analyses of viral mutational pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although protein receptors on the plasma membrane involved in the initial steps of productive HIV-1 infection have been well characterized, little is known about interactions between cellular carbohydrate receptors and HIV-1. Here, we report the involvement of a carbohydrate receptor, the macrophage mannose receptor (MR), and its role in supporting HIV-1 binding and entry. HIV-1 can enter the cytoplasm of human macrophages and microglia as well as murine macrophages by MR, although no subsequent viral replication was observed. Correspondingly, HIV-1 entry into Cos-7 cells after induction of expression of MR by transfection with MR-cDNA did not demonstrate viral replication. Our studies suggest that whereas MR may serve as a binding and an entry site, the MR-mediated pathway does not lead to productive HIV-1 infection. In addition, we report that recombinant HIV-1 gp120 blocks MR-mediated phagocytosis in human and murine alveolar macrophages and microglial cells. Therefore, characterization of the HIV-1 noninfectious MR-mediated phagocytic pathway may foster advances in HIV-1 vaccine design and an improved understanding of HIV-1/AIDS pathogenesis and host defenses.

We investigated the interactive relationship between proviral DNA load and virus-specific IFN-gamma-secreting T cell responses in HIV-1C infection. The presence or absence of correlation, and inverse or direct type of correlation, if any, were dependent on targeted viral gene product. Responses to Gag p24 or to Pol were associated with lower proviral DNA load. Associations between proviral DNA load and T cell responses did not necessarily mirror relationships between plasma RNA load and T cell responses. An interaction analysis showed a synergy in that lower proviral DNA and lower plasma RNA load were associated with high Gag p24-specific IFN-gamma-secreting T cell response (interaction test P = 0.0003). Our findings support the idea that HIV proteins have differential value for vaccine design, and suggest that, for HIV-1C, Gag p24 may be one of the most attractive regions to include in vaccine designs to control both plasma RNA load and cell-associated proviral DNA load.

Human immunodeficiency virus type 1 subtype C (HIV-1C) accounts for about 50% of all HIV infections in the pandemic and is the predominant subtype in the heavily burdened region of southern Africa. HIV-1C possesses unique genetic and phenotypic features that might be associated with biological differences compared to other subtypes. Here, we generated virus isolates from individuals at different stages of HIV-1C infection and investigated the chemokine receptor repertoire that the derived HIV-1C isolates may utilize for entry. Our results show that the R5 phenotype predominates among viruses in Botswana, with a lesser contribution of viruses showing the dualtropic X4R5 phenotype. No viruses of pure X4 phenotype were found, which suggests no discernable evolution of HIV-1C to a monotropic X4 phenotype as the epidemic ages in Botswana. Usage of other coreceptors was rare and apparently insignificant. These results enhance our understanding of HIV-1C biology, with implications for designing and testing therapeutic and prophylactic agents.

Virus-specific T-cell immune responses are important in restraint of human immunodeficiency virus type 1 (HIV-1) replication and control of disease. Plasma viral load is a key determinant of disease progression and infectiousness in HIV infection. Although HIV-1 subtype C (HIV-1C) is the predominant virus in the AIDS epidemic worldwide, the relationship between HIV-1C-specific T-cell immune responses and plasma viral load has not been elucidated. In the present study we address (i) the association between the level of plasma viral load and virus-specific immune responses to different HIV-1C proteins and their subregions and (ii) the specifics of correlation between plasma viral load and T-cell responses within the major histocompatibility complex (MHC) class I HLA supertypes. Virus-specific immune responses in the natural course of HIV-1C infection were analyzed in the gamma interferon (IFN-gamma)-enzyme-linked immunospot assay by using synthetic overlapping peptides corresponding to the HIV-1C consensus sequence. For Gag p24, a correlation was seen between better T-cell responses and lower plasma viral load. For Nef, an opposite trend was observed where a higher T-cell response was more likely to be associated with a higher viral load. At the level of the HLA supertypes, a lower viral load was associated with higher T-cell responses to Gag p24 within the HLA A2, A24, B27, and B58 supertypes, in contrast to the absence of such a correlation within the HLA B44 supertype. The present study demonstrated differential correlations (or trends to correlation) in various HIV-1C proteins, suggesting (i) an important role of the HIV-1C Gag p24-specific immune responses in control of viremia and (ii) more rapid viral escape from immune responses to Nef with no restraint of plasma viral load. Correlations between the level of IFN-gamma-secreting T cells and viral load within the MHC class I HLA supertypes should be considered in HIV vaccine design and efficacy trials.

A systematic analysis of immune responses on a population level is critical for a human immunodeficiency virus type 1 (HIV-1) vaccine design. Our studies in Botswana on (i) molecular analysis of the HIV-1 subtype C (HIV-1C) epidemic, (ii) frequencies of major histocompatibility complex class I HLA types, and (iii) cytotoxic T-lymphocyte (CTL) responses in the course of natural infection allowed us to address HIV-1C-specific immune responses on a population level. We analyzed the magnitude and frequency of the gamma interferon ELISPOT-based CTL responses and translated them into normalized cumulative CTL responses. The introduction of population-based cumulative CTL responses reflected both (i) essentials of the predominant virus circulating locally in Botswana and (ii) specificities of the genetic background of the Botswana population, and it allowed the identification of immunodominant regions across the entire HIV-1C. The most robust and vigorous immune responses were found within the HIV-1C proteins Gag p24, Vpr, Tat, and Nef. In addition, moderately strong responses were scattered across Gag p24, Pol reverse transcriptase and integrase, Vif, Tat, Env gp120 and gp41, and Nef. Assuming that at least some of the immune responses are protective, these identified immunodominant regions could be utilized in designing an HIV vaccine candidate for the population of southern Africa. Targeting multiple immunodominant regions should improve the overall vaccine immunogenicity in the local population and minimize viral escape from immune recognition. Furthermore, the analysis of HIV-1C-specific immune responses on a population level represents a comprehensive systematic approach in HIV vaccine design and should be considered for other HIV-1 subtypes and/or different geographic areas.

An evolving dominance of human immunodeficiency virus type 1 subtype C (HIV-1C) in the AIDS epidemic has been associated with a high prevalence of HIV-1C infection in the southern African countries and with an expanding epidemic in India and China. Understanding the molecular phylogeny and genetic diversity of HIV-1C viruses may be important for the design and evaluation of an HIV vaccine for ultimate use in the developing world. In this study we analyzed the phylogenetic relationships (i) between 73 non-recombinant HIV-1C near-full-length genome sequences, including 51 isolates from Botswana; (ii) between HIV-1C consensus sequences that represent different geographic subsets; and (iii) between specific isolates and consensus sequences. Based on the phylogenetic analyses of 73 near-full-length genomes, 16 "lineages" (a term that is used hereafter for discussion purposes and does not imply taxonomic standing) were identified within HIV-1C. The lineages were supported by high bootstrap values in maximum-parsimony and neighbor-joining analyses and were confirmed by the maximum-likelihood method. The nucleotide diversity between the 73 HIV-1C isolates (mean value of 8.93%; range, 2.9 to 11.7%) was significantly higher than the diversity of the samples to the consensus sequence (mean value of 4.86%; range, 3.3 to 7.2%, P < 0.0001). The translated amino acid distances to the consensus sequence were significantly lower than distances between samples within all HIV-1C proteins. The consensus sequences of HIV-1C proteins accompanied by amino acid frequencies were presented (that of Gag is presented in this work; those of Pol, Vif, Vpr, Tat, Rev, Vpu, Env, and Nef are presented elsewhere [http://www.aids.harvard.edu/lab&#95;research/concensus&#95;sequence.htm]). Additionally, in the promoter region three NF-kappa B sites (GGGRNNYYCC) were identified within the consensus sequences of the entire set or any subset of HIV-1C isolates. This study suggests that the consensus sequence approach could overcome the high genetic diversity of HIV-1C and facilitate an AIDS vaccine design, particularly if the assumption that an HIV-1C antigen with a more extensive match to the circulating viruses is likely to be more efficacious is proven in efficacy trials.

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.

Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells., (Copyright 2000 Academic Press.)

A nearly full-length genome sequence of a novel HIV-1 A/J recombinant with a complex structure of the pol gene has been analyzed. This virus was isolated in 1998 from a 35-year-old female from Botswana. The virus demonstrated a dual pattern for CXCR4/CCR5 coreceptor utilization. Using short-term enrichment of the donor's PBMCs, the 98BW21 isolate was long-range amplified, cloned, and sequenced. The sequence of the clone 98BW21.17 spanned 9103 bp from the PBS site to the U5 region of the 3' LTR. The phylogenetic relationship of the 98BW21.17 clone to HIV-1 sequences represented by M, N, and O groups and A-K subtypes of the M group was examined across the entire viral genome. The 98BW21.17 clone demonstrated a unique phylogenetic topology clustering within subtype A or subtype J reference sequences. However, the subtype origin of two regions within the pol gene (p51 RT and integrase) could not be identified. Recombination patterns of the 98BW21.17 clone were different from known AGJ/AGIJ-type viruses such as isolates BFP90 and 95ML84. This study demonstrated the existence and replication competence of a new dual-tropic X4/R5 recombinant form of HIV-1 on the subtype J backbone. The nucleotide sequence of the 98BW21.17 clone was submitted to GenBank under accession number AF192135.

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9. 1% versus 6.5 and 4.3%, respectively; P < 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.

Differential rates of AIDS development and/or T4 lymphocyte depletion in HIV-1-infected individuals remain unexplained. The hypothesis that qualitative differences in selection pressure in vivo may account for different rates of disease progression was addressed in nine eligible study participants from a cohort of 315 homosexual men who have been followed since 1985. Disproportionately fewer changes in variable regions and more in C3 of gp12O were found to be significantly associated with slower disease progression. Our finding provides the first example to demonstrate that differential selection pressure related to the emergence of HIV-1 variants is associated with long term nonprogression. Candidate vaccines that elicit strong selection pressure on C3 of gp120 are likely to provide better protection than those targeting variable regions.

The antibody response to the HIV-1 envelope protein has not been well characterized in patients with AIDS dementia complex (ADC). We evaluated the frequency of antibodies against the HIV-1 envelope in cerebrospinal fluid (CSF) and serum from 21 persons with ADC and 10 symptom-free HIV-1-positive subjects using Western immunoblot with reducing and nonreducing buffer and radioimmunoprecipitation (RIP) analysis. RIP analysis revealed anti-envelope antibodies in all sera tested. Higher anti-envelope levels were observed in CSF than in serum of 12 of 21 ADC patients and only 1 of 10 symptom-free subjects (two-sided Fisher exact test, p < 0.05). All persons with moderate to severe ADC had higher anti-envelope levels in CSF than in sera (p < 0.005). CSF anti-gp120 antibodies were not as readily detected by Western blot analysis even under nonreduced conditions, suggesting that they are directed to conformational epitopes. Higher CSF anti-envelope antibodies appear to be more common in patients with ADC than in symptom-free HIV-1-positive subjects. This antibody pattern may serve as a marker for ADC and its progression.

Heterosexual transmission by vaginal intercourse accounts for most transmission of human immunodeficiency virus-type 1 (HIV-1) in Africa and Asia but is less important in the HIV-1 epidemics of the United States and Western Europe. Epithelial Langerhans' cells (LCs) represent a possible source of initial cell contact for vaginal infection. Fifteen primary isolates of HIV-1 from U.S. homosexuals and 18 HIV-1 isolates from Thailand heterosexuals were evaluated for growth in LCs of U.S. origin. All the viruses from the Thai heterosexuals, which were subtype E, grew more efficiently in the LCs than any of the viruses from the U.S. homosexuals, which are subtype B. These results suggest that LC tropism is associated with the efficiency of heterosexual transmission of HIV.

Virus-specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV-1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)-7 can affect T cell maturation and differentiation. Here we report on a group of five HIV-1-positive individuals who tested negative for env- and gag-specific CTL activity. When exogenous recombinant human IL-7 was added as a stimulus to the cultures, none (0/5) of the CTL-negative individuals exhibited a CTL response. Individuals that were negative for HIV-1-specific CTL activity were found to lack IL-7 receptor (IL-7R) on CD8+ cells with a comparable reduction on CD4+ cells. Increased shedding of IL-7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of 125I-labeled IL-7 and Scatchard analysis. The lack of IL-7R is probably not due to endogenous IL-7, since it was not detectable in the culture supernatants of the patients studied. HIV-1 proteins may cause down-modulation of IL-7R expression, either by producing an insufficient number of molecules or by rapid decay of IL-7R on T cells. These changes may alter the cells' capability to respond to the IL-7 growth signal, resulting in CTL failure and subsequent mishandling of the virus.

Understanding the mechanism by which human immunodeficiency virus type 1 (HIV-1) kills CD4+ T lymphocytes is important to the development of therapeutic and prophylactic strategies. Recent studies have indicated that, in some cases, progression to AIDS is associated with the appearance of syncytium-inducing, T cell line-tropic HIV-1 variants. Nevertheless, approximately 50% of subjects with AIDS harbor only non-syncytium-inducing, macrophage-tropic (NSI-M) variants of HIV-1. In most asymptomatic patients, NSI-M HIV-1 isolates are the predominant virus type found. We report here that cytopathicity of NSI-M HIV-1 for primary CD4+ T lymphocytes can be directly detected in vitro. The extent of CD4+ T-cell killing was not completely correlated with the rate of viral replication, suggesting that other characteristics of HIV-1 contribute to its cytopathicity. Our findings suggest that: (i) direct killing by NSI-M HIV-1 may contribute to CD4+ T-lymphocyte depletion in vivo, and (ii) the determinants of HIV-1 cytopathicity for CD4+ T lymphocytes and cell tropism or syncytia-forming ability are not necessarily tightly linked.

A high frequency of autoantibodies to brain proteins has been reported in HIV-1-positive patients. However, the specificity of this response has not been characterized. Using homogenized tissue from three normal brains, the presence of autoantibodies to human brain proteins was analyzed in 16 HIV-1-positive patients with AIDS dementia complex (ADC), 10 HIV-1-positive patients without ADC, 10 patients with multiple sclerosis, 10 patients with juvenile rheumatoid arthritis, and 10 normal controls. Although antibodies to various brain proteins were detected in sera from one-third HIV-1-infected individuals with or without ADC, the proteins recognized were different among different brains. Only one ADC patient had consistent seroreactivity to a 50-kDa brain-specific protein. Our results indicate that autoantibodies to brain proteins are infrequently present in patients with ADC.

Immunologically cross-reactive proteins in the human brain that resemble the V3 loop of human immunodeficiency virus type 1 (HIV-1) gp120 have been identified. When several homogenized tissues from normal brains were used, a monoclonal antibody raised against amino acids 308 to 320 of the V3 loop reacted with three prominent human brain proteins (HBP) of 35, 55, and 110 kDa. Among the three, the 55-kDa HBP appears to be specific to the central nervous system. These results indicate that the V3 loop of HIV-1 gp120 shares an epitope with HBP. An immune response to the V3 loop that generates cross-reactive antibodies to cellular proteins may be an autoimmune mechanism by which HIV-1 can damage the central nervous system.

In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp41). Truncation of 147 amino acids from the carboxyl terminus of gp41 (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp41 generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp41 required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp41 did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp41 also plays a role in other steps of virus replication.

T-lymphotropic retroviruses of cats cause lymphopenia and immunosuppression and represent the major cause of death in that species. Similarly HTLV-I which is T4 tropic is associated with an increased risk for development of infectious disease in regions where the virus is endemic. Since HTLV-I is also believed to be transmitted by blood and by sexual intercourse we considered the possibility that a variant form of HTLV might cause AIDS. The identification of cross-reactive antibodies to HTLV-I-MA in a third or more of the AIDS patients and in suspicious blood donors that donated to transfusion-associated cases of AIDS eventually led to the recognition of HTLV-III, the causative agent of AIDS. The protein most associated with lymphocyte immortalization or transformation in the case of HTLV-I is p42. The proteins of HTLV-I encoded by the amino terminus of the env gene designated gp61 and gp45 are the most immunogenic antigens of this virus. Similarly those encoded by the amino terminus of the env gene HTLV-III designated gp160 and gp120 appear to be the most immunogenic markers for this agent. Almost all AIDS patients, ARC patients, and asymptomatic hemophiliacs have detectable antibodies to gp120 and gp160. HTLV-III related agents designated STLV-III have been found in macaque monkeys that develop simian AIDS and high prevalence rates of antibodies to STLV-III can be found in healthy African green monkeys. We hypothesize that the STLV-III of African green monkeys could represent a recent source of the virus to have infected humans in central Africa where the human epidemic probably began. The recognition that up to one million people may already be infected with HTLV-III in the United States alone indicates the need for development of a vaccine. The availability of primate species infected with the serologically related STLV-III agents that either resist disease development (African green monkeys) or succumb to an AIDS-type syndrome (rhesus) provide models that should aid in our attempts to develop such vaccines.

Of 96 patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex and healthy individuals at risk for AIDS, 4 had no detectable antibodies to viral proteins, though human T-cell leukaemia (lymphotropic) virus type III was isolated from their lymphocytes. 3 of these subjects were symptom-free and 1 had lymphadenopathy. All 4 were sexual partners of patients with AIDS or AIDS-related complex. The occurrence of seronegative but virus-positive persons without clinical symptoms suggests that assays other than those detecting antibody to virus, perhaps based on detection of viral antigens or immune complexes, may be required to identify all infected individuals.

Antibodies from the serum of patients with the acquired immune deficiency syndrome (AIDS) or with the AIDS-related complex and from the serum of seropositive healthy homosexuals, recognize two major glycoproteins in cells infected with human T-cell lymphotropic virus type III (HTLV III). These glycoproteins, gp160 and gp120, are encoded by the 2.5-kilobase open reading frame located in the 3' end of the HTLV-III genome, as determined by amino terminus sequence analysis of the radiolabeled forms of these proteins. It is hypothesized that gp160 and gp120 represent the major species of virus-encoded envelope gene products for HTLV-III.

An assay for antibodies to membrane antigens of cells infected by human T-cell leukemia virus was used to examine serum from persons who donated blood to 12 patients with acquired immunodeficiency syndrome (AIDS) associated with blood transfusions. The occurrence of positive results in the assay was significantly greater among donors to the AIDS patients (9 of 117; 7.7 percent) than among random donors (1 of 298; 0.3 percent). Of 12 sets of donors examined, 9 sets included a donor whose serum gave positive results for the presence of the antibodies. In six of these nine sets, the seropositive donor was an individual who was also identified as a possible source of AIDS transmission when epidemiologic and immunologic criteria were used.

Specific antibodies to cell membrane antigens found on human T-cell leukemia virus (HTLV)-infected cells have been detected in Japanese patients with adult T-cell leukemia/lymphoma and in asymptomatic carriers, using a live cell-membrane immunofluorescence assay. Reactivity of the positive antisera was analyzed using radioimmunoprecipitation and NaDodSO4/PAGE with the HTLV-infected tumor cell line Hut 102 (clone B2). The major cell-associated antigens identified include two glycoproteins of approximately equal to 61 and 45 kDa, which appear to be the most immunogenic species in exposed people, a nonglycosylated species of 42 kDa, and four additional species that contain gag gene-encoded antigens with sizes ranging from 19 to 55 kDa. The two glycoproteins ( gp61 and gp45 ) are encoded, at least in part, by the env gene of HTLV as evidenced by amino acid sequence analysis.

People exposed to type I human T-cell leukemia virus (HTLV-I) develop antibodies to an antigen at the surface of virus-infected cells, designated human T-cell leukemia virus membrane antigen (HTLV-MA). In an earlier study, we demonstrated that the major component of HTLV-MA is gp61, a glycoprotein encoded by the HTLV env gene. In the current study, we found that human antibodies that react with HTLV-MA on cells infected with HTLV-I react equally well with HTLV-MA on C3-44/MO, a target cell infected with type II HTLV. A glycoprotein with an approximate size of 67 kDa, gp67, was identified in C3-44/MO using immunoprecipitation and NaDodSO4/PAGE analysis. The positions of serine and cysteine residues were determined in the amino terminus of gp67 by radiolabel sequencing analysis. Comparison with the amino acid sequence deduced from the primary nucleotide sequence of HTLV-IIMO virus reveals that gp67 is also encoded, at least in part, by the env gene. The gp67 of HTLV-IIMO, like the env gene product of HTLV-ICR, gp61, is recognized both by antibodies from a HTLV-IIMO-infected patient with a variant form of hairy cell leukemia, and by antibodies from patients with HTLV-I-associated adult T-cell leukemia/lymphoma. These results indicate that, despite the divergence between HTLV-I and HTLV-II, the major env gene products of the two types of HTLV are conserved to the degree that they are serologically cross-reactive.

Along with homosexual men, Haitians, and intravenous drug abusers, hemophiliacs are at high risk of contracting acquired immunodeficiency syndrome (AIDS). An earlier study revealed that 36 percent of a group of the AIDS patients had antibodies to cell membrane antigens associated with the human T-cell leukemia virus (HTLV-MA), whereas only 1.2 percent of matched asymptomatic homosexual controls had these antibodies. In the present experiments, serum samples from 172 asymptomatic hemophiliacs were examined for the presence of antibodies to HTLV-MA. Such antibodies were detected in 5 to 19 percent of the hemophiliacs examined from four geographical locations, but in only 1 percent or less of laboratory workers, normal blood donors, donors on hemodialysis, or donors with chronic active hepatitis.

This report describes serologic evidence for a virus similar to that known as simian T-lymphotropic virus type III of African Green monkeys (STLV-IIIAGM) infecting apparently healthy people in Senegal, West Africa, and the isolation of virus from these individuals. Serum samples from selected healthy West African people showed unusual serologic profiles when tested with antigens of HTLV-III/LAV, the etiologic agent of AIDS, and of STLV-IIIAGM. The samples reacted strongly with all of the major viral antigens of STLV-IIIAGM but showed variable or no reactivity with the major viral antigens of HTLV-III/LAV by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A new human T-lymphotropic virus (HTLV-IV) isolated from these people was grown in vitro and shown to have retroviral type particles, growth characteristics, and major viral proteins similar to those of the STLV-III and HTLV-III/LAV group of retroviruses. The gp120/160, gp32, p64, p55, p53, p24, and p15 proteins precipitated were the same size as and reactive with STLV-IIIAGM proteins. The serologic data suggest that this virus shares more common epitopes with STLV-IIIAGM than with the prototype HTLV-III/LAV that infects people in the United States and Europe. Further study of this virus and of the origin of the HTLV-III/LAV group of viruses may expand our understanding of the human AIDS virus.

The most variable proteins, the gp120's, of the many isolates of HIV-I can be readily compared by two-dimensional oligopeptide maps. The gp120 in a given cell line is completely stable, but the cell line defines the actual gp120 size and may induce minor peptide changes. HTLV-IIIB and LAV differ slightly from each other even when grown in the same cell line, while LAV grown in a B cell line is less related. Molecularly distant isolates have unique patterns. While anti-HTLV-IIIB gp120 antibody neutralized both HTLV-IIIB and LAV, it recognizes only the homologous HTLV-IIIB infected cells in cytotoxicity assays. Structural analysis of isolates should be helpful in defining the range of immunological reactivities among variants as a contribution to a rational approach to a vaccine against AIDS.